Yeast Vacuolar Dynamics Visualization

Pulse-chase labeling of log phase yeast with vacuolar fluorescent dye FM4-64 and fluorescence microscopy imaging were performed as described (Nickerson et al. 2012 (link)), except that cells were grown in nonselective YPD prior to labeling and imaging. DNA in agarose gels was stained using ethidium bromide and visualized using ultraviolet light in a BioRad Chemi-doc system with digital camera and Quantity One imaging software (BioRad, Hercules, CA, USA). All gel images were exported from Quantity One as .TIFF images, except Figure 2B, which was printed to photographic paper and later scanned in .TIFF format using an Epson flatbed scanner. Images were cropped using Adobe Photoshop CS6 (Adobe, San Jose, CA, USA). Fluorescence microscopy images were overlaid using ImageJ (NIH, https://imagej.nih.gov/ij/). Images were arranged as annotated figures using Canvas Draw 4 vector graphics software (Canvas GFX, Boston, MA, USA).